Transcript eau d’e coli igem 2006: mit The Undergrads: meet the team Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological Engineering Veena Venkatachalam Chemistry / Physics Boyuan Zhu Electrical.

eau d’e coli
igem 2006: mit
The Undergrads:
meet the team
Kate Broadbent
Biological Engineering
Andre Green
Biology / Physics
Stephen Payne
Biological Engineering
Veena Venkatachalam
Chemistry / Physics
Boyuan Zhu
Electrical Engineering and
Computer Science
The Grad Students:
Barry Canton
Austin Che
Jason Kelly
Reshma Shetty
Samantha Sutton
The Faculty:
Drew Endy
Tom Knight
motivations
eau d’e
coli
mit igem
2006
background
A. thaliana
O.
A. majus C. breweri
basilicum
S.
cerevisiae
JMT
CCMT
BAMT
BSMT
ATF1
jasmine
cinnamon
fresh / floral
wintergreen
banana
background
O.
A. majus C. breweri
basilicum
S.
cerevisiae
CCMT
BAMT
BSMT
ATF1
cinnamon
fresh / floral
wintergreen
banana
background
A. majus C. breweri
S.
cerevisiae
BAMT
BSMT
ATF1
fresh / floral
wintergreen
banana
background
C. breweri
S.
cerevisiae
BSMT
ATF1
wintergreen
banana
timeline:
initial planning
initial planning • scent production • self-generating scent • production regulation • eliminating indole
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
timeline:
scent production
initial planning • scent production • self-generating scent • production regulation • eliminating
indole
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
timeline:
self-generating scent
initial planning • scent production • self-generating scent • production regulation • eliminating indole
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
timeline:
production regulation
initial planning • scent production • self-generating scent • production regulation • eliminating indole
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
timeline:
indole elimination
initial planning • scent production • self-generating scent • production regulation • eliminating
indole
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
wintergreen generating
device
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
device generates
wintergreen
E. coli
no methyl salicylate
[no peak at 9.1
minutes]
E. coli + precursor
(SA) + WGD
methyl salicylate
MW = 152
O
O
OH
banana generating
device
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
precursor biosynthesis
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
precursor biosynthesis
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
goal: the regulated
system
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
growth dependent
regulation
osmY: active in stationary phase & under high osmotic pressure conditions
osm
Y
osmY + inverter
regulation is growth
dependent
2E+5
BBa_J45995 (pOsmY-GFP)
GFP Signal (relative units)
2E+5
BBa_J45017 (Negative control)
2E+5
BBa_I7100 (Constitutive promoter-GFP)
1E+5
1E+5
1E+5
8E+4
6E+4
4E+4
2E+4
0E+0
0.0
0.1
0.2
0.3
0.4
Absorbance 600nm (relative units)
0.5
regulation is growth
dependent
regulated system:
exponential
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
regulated system: stationary
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
regulated system
chassis (tnaA5-) does not produce
indole
wild type E. coli
chassis: tnaA5- E. coli
Indole
Peak at 10 minutes
MW = 117
No indole
[no peak at 10
minutes]
final system
smell test results
100
90
80
Percentage
70
60
Banana
M int
Stinky
50
40
30
20
10
0
A (Mint)
B (Stinky)
C (Banana)
future applications
Improve the workplace environment for
microbiologists working with E. coli
Port our system to bacterial species involved in
bioremediation.
Implement our system in bacteria responsible
for bad human odor in the mouth, armpits and
feet.
Implement our system in yeast used in bread
and beer production
conclusion
Designed, built, and tested a synthetic
biological system over the course of
summer
Engineered synthetic scent systems in E.
coli
Regulated via transcription control devices
acknowledgments
Natalia Dudareva, Purdue University: gifts of expression vectors encoding BAMT,
SAMT and BSMT enzymes.
Eran Pichersky, University of Michigan: suggestion of eliminating indole from
Escherichia coli to mitigate the natural "bad" smell.
Mary Berlyn, CGSC, The Coli Genetic Stock Center: Esherichia coli strain YYC912.
Brian Cook, MIT: valuable discussions.
Cornelia Reimmann, University of Lausanne: gift of an expression vector carrying the
pchBA coding region.
Dieter Haas, University of Lausanne: gift of the Pseudomonas fluorescens strain
CHA0.
Peter Bakker, Utrecht University, The Netherlands: gift of an expression vector
carrying the pmsCEAB coding region, as well as, gift of the Pseudomonas fluorescens
strain WCS374.
Herbert P. Schweizer, Colorado State University: gift of the pUCP22 Esherichia coli to
Pseudomonas shuttle vector.
Pamela Silver, Harvard Medical School: gift of yeast BioBricks vector.
questions and
answers
growth curves
Absorbance 600nm (relative units)
0.70
0.60
0.50
0.40
0.30
0.20
Bba_J45995 (pOsmY-GFP)
0.10
Bba_J45017 (Negative control)
BBa_I7100 (Constitutive promoter-GFP)
0.00
0
10000 20000 30000 40000 50000 60000 70000
Time (min)